Tumors are embedded into a surrounding microenvironment that consists of extracellular matrix and non- malignant body cells. The microenvironment strongly affects tumor progression by releasing factors that influence tumor growth, cancer stem cell function and metastasis. Identifying these factors and understanding their underlying molecular mechanism is crucial for the development of therapeutic approaches to treat cancer patients. Matrix Metalloproteinases (MMPs) have been implicated in breast cancer for many years and they have been considered as therapeutic targets to treat cancer patients. However, previous clinical cancer trials using synthetic small molecule inhibitors to interfere with MMP catalytic activity yielded disappointing results. My postdoctoral research has now shown that MMPs such as MMP3 may work in a non-proteolytic manner through their hemopexin domain. This may explain why compound inhibitors targeting the catalytic function of MMPs failed to improve disease outcome. In this proposal, I will explore the molecular mechanism underlying the role of MMP3 in breast cancer and ask whether the MMP3 hemopexin domain may serve as a therapeutic target in breast cancer. During the mentored phase, I will learn how to utilize mouse models to study breast cancer stem cells as well as methods to monitor the Wnt signaling pathway to investigate the function of MMP3 in breast cancer. During my postdoc, I have established that MMP3 modulates the Wnt signaling pathway by binding and inactivating Wnt5b via the hemopexin domain and that this plays an important role in stem cell maintenance during normal homeostasis of the mammary epithelium. I will use the skills I learn during the mentored phase to determine whether this interaction of MMP3 with Wnt5b also affects breast cancer progression. In particular I will employ lentiviral transduction, transplantation of mammary epithelial cells, mouse models of breast cancer in combination with tools to monitor Wnt signaling to address whether (1) MMPs regulate breast cancer stem cells through Wnt signaling, and whether (2) non-canonical Wnt signaling mediated by Wnt5b is involved in the regulation of stem cells during normal homeostasis and in breast cancer. These projects will provide the rationale to ask whether my results can be translated into possible therapeutic application by specifically targeting the MMP3-hemopexin domain. This will be the goal of aim 3 during the independent phase of this grant. These experiments include lentiviral overexpression of only the hemopexin domain in normal mammary epithelial cells and breast cancer cell lines to test whether the hemopexin domain is sufficient for tumor initiation and progression. I will also specifically target the hemopexin domain using antibodies or small compound inhibitors. Taken together, these studies will expand our knowledge about the regulation of stem cell biology in normal homeostasis and in cancer. My results can explain why previous clinical trials using MMP compound inhibitors targeting the catalytic, but not the hemopexin domain of MMPs, performed poorly in treating cancer patients. Ultimately, this may provide the rationale for the development of novel therapeutic approaches to treat patients suffering from breast cancer.